In order to test the granule exocytosis model for lymphocyte cytotoxicity, we have examined the cytotoxic activity of the rat mucosal mast cell tumor line RBL after transfection with genes for cytotoxic lymphocyte granule components. We have constructed triple, double, and single RBL transfectants expressing cytolysin (cy) and the granule serine proteases granzyme A (gza) and granzyme R (gzb). RBL-cy transfectants show only modest cytotoxicity on tumor targets, with no accompanying target DNA degradation. While RBL-gza transfectants express gza at levels comparable to cloned CTL and secrete it in response to IgER cross-linking, they have no cytotoxic activity detectable. RBL-cy-gza transfectant clones showing good expression of both these granule components showed cytolytic activity comparable to RBL-cy on RBC targets, but were greater than 3x more lytic on three different tumor targets. This cytotoxicity is accompanied by target DNA fragmentation. To confirm that killer cell granzymes need to enter the target cell, we loaded target cells with the macromolecular protease inhibitor aprotinin by osmotic lysis of pinosomes. Compared to BSA-loaded targets or unloaded targets, aprotinin-targets were less susceptible to lysis and DNA breakdown by CTL and RBL transfectants expressing granzyme A. However, RBL transfectants expressing only cytolysin lysed BSA-loaded and aprotinin-loaded targets with equal efficiency. As a direct test of the ability of proteases to induce cytotoxicity when introduced into the cytoplasm of a target cell, we have "injected" various proteases into tumor cells using osmotic lysis of pinosomes. The endoproteases trypsin, chymotrypsin, and proteinase K were all found to lyse several different types of tumor cells in a dose dependent manner, as measured by 51Cr release. This death was generally apoptotic by morphological criteria and DNA fragmentation.